MicroRNAs are 17-24 nucleotide-long, single-stranded RNA molecules that profoundly impact the gene expression program of a cell by annealing to and suppressing the expression of many target genes. Changes in expression of specific microRNAs and other types of short RNAs are involved in different types of malignancies, including prostate cancer. We know that progression of prostate cancer is accompanied by changes in the gene expression program, but the role of microRNA and other short RNAs in progression have not been studied. In this proposal we will clone and sequence Solexa/lllumina libraries of small RNAs from prostate cancer cells to identify microRNAs and other short RNAs that are (a) regulated by PTEN loss, (b) regulated by overexpression of PKN1 and (c) correlated with progression to androgen independence. We will also follow the changes in known microRNAs by hybridization to locked-nueleic acid microarrays. These results will identify new short RNAs involved in prostate cancer progression and test the hypothesis that specific molecular genetic lesions are correlated with specific microRNA changes during the progression of prostate cancer. We will determine whether microRNAs repressed during prostate cancer progression contribute to the phenotype of progression, both in vitro and in vivo, starting with four microRNAs (in two families) that have already been validated in preliminary results. Finally, we will identify targets of these microRNAs whose de-repression during cancer progression leads to worsening ofthe malignant phenotype. We will identify the targets of two key microRNA families involved in cancer progression by a novel combination of experiments and computational target prediction, starting with eight targets that have already passed the experimental filters. Thus new technologies, short RNA cloning and ultrahigh-throughput sequencing, and new combinations of assays for identification of relevant targets will be applied to the molecular analysis of prostate cancer progression, and the function of key microRNAs In this process will be dissected. The results are expected to open new avenues of research, therapy, and diagnosis of prostate cancers.